Transplantation of insulin-producing cells in many diabetes patients potentially would restore normal glucose homeostasis and prevent the severe long-term complications of the disease. However, the scarcity of donated pancreata currently limits transplantation of the whole organ or of isolated pancreatic islets to a tiny fraction of those patients who might benefit from such treatment. Directed differentiation of stem cells offers a possible means to generate abundant insulin-producing cells. A promising new source of stem cells has been identified, namely, amniotic fluid collected for prenatal genetic testing. Amniotic fluid-derived stem (AFS) cells can be expanded extensively in culture, do not form teratoma tumors, and have the capacity to yield a variety of specialized cell types. Preliminary experiments with mouse AFS cells showed that they can give rise to insulin-producing cells resembling beta-cells of the pancreas. Differentiation along this lineage was promoted by transient expression of the pancreatic transcription factor Pdx-1. The proposed project will determine whether human AFS cells, driven by Pdx-1 provided from an expression vector, can similarly yield insulin-producing cells of the pancreatic lineage. Two (2) complementary strategies will be explored to develop a stem cell-based therapy for diabetes. 1 approach will be to generate clusters of insulin-producing cells in culture that resemble pancreatic islets ("neo-islets") and could be utilized in a transplantation procedure that has proven successful with isolated islets, the Edmonton Protocol. Human AFS cells will be induced to express human Pdx-1 by introduction of a plasmid vector via nucleofection, a high efficiency form of electroporation. The cells will be cultured in a 2-stage system shown previously to support the production from mouse AFS cells, transduced with a Pdx-1 vector, of insulin-producing cells in neo-islet structures. The resulting differentiated human cells will be assayed for multiple markers of pancreatic beta-cells and for the capacity to synthesize insulin and to secrete it in a glucose-responsive manner. The potential therapeutic value of neo-islets produced from human AFS cells will be tested by transplantation under the kidney capsule in immune-deficient mice rendered diabetic by streptozotocin, a toxin that destroys endogenous pancreatic beta-cells. The second approach will be to introduce the Pdx-1 expression vector into the human stem cells and then inject them directly into the circulation of diabetic mice. The treated animals will be tested for restoration of normal regulation of blood glucose, the production of human-specific insulin and C-peptide, and the regeneration of pancreatic islets by the human cells. Preliminary studies with mouse AFS cells suggest that this approach can provide long-term reversal of diabetes. [unreadable] [unreadable] [unreadable]